Benzo[a]pyrene uptake into rat liver microsomes: Effects of adsorption of benzo[a]pyrene to asbestos and non-fibrous mineral particulates

The fluorescence yield of benzo[a]pyrene (BP) increases dramatically upon its transfer from the surface of particulates to rat liver microsomes. Adsorption of BP to Canadian chrysotile, anthophyllite, hematite and silica results in greatly enhanced uptake rates into microsomes when compared to uptake from a microcrystalline dispersion of BP. The fibrous minerals chrysotile and anthophyllite were more effective than silica and hematite in enhancing BP uptake. Simple mixtures of BP microcrystals and particles did not display enhanced transport, indicating that adsorption of BP to the particulate surface is necessary for enhanced microsomal uptake. BP was not released into microsomes from carbon black.We suggest that particulate-enhanced availability of BP may be of significance in the co-carcinogenesis between particulates and polynuclear aromatic hydrocarbons. However, other mechanisms are also possible, and are not excluded by our experiments. The fluorescence methodology described in this paper provides a novel and convenient means to quantify microsomal uptake of BP and thereby investigate further the mechanisms of cocarcinogenesis.     

Degradation of Benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Benzo[a]pyrene removal by Marasmiellus troyanus in soil microcosms

Benzo[a]pyrene (B[a]P) is a carcinogenic polyaromatic hydrocarbon that enters the environment as an incomplete combustion production of fossil fuels. Several species of filamentous fungi are capable of biotransforming and/or mineralizing B[a]P in liquid cultures, however there has been less success in soil habitats. In this study, the litter rot fungus Marasmiellus troyanus was encapsulated in alginate and delivered to B[a]P-spiked soil microcosms (100 μg B[a]P/g soil) for 1, 2 and 6 weeks, with and without a fertilizer solution. After 2 weeks, 32.5% of B[a]P was recovered from soil microcosms treated with M. troyanus compared to 55–70% for controls. After 6 weeks, controls demonstrated an average percent recovery of B[a]P of 54% while M. troyanus-inoculated samples gave an average percent recovery of 11%. Similar bioaugmentation of contaminated habitats with appropriately formulated fungi has potential for practical bioremediation in soil environments. Journal of Industrial Microbiology & Biotechnology (2000) 25, 116–119.     

Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor

The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of <=7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rot fungi.     

Removal of Benzo[a]pyrene by a fungus Aspergillus sp. BAP14

A fungal strain BAP14 isolated from marine sediments of coast in Xiamen city, was found to have the ability to degrade benzo[a]pyrene (BaP), and identified as Aspergillus sp. based on 18S rRNA gene sequence. Aspergillus sp. BAP14 was able to remove about 30 and 60% of BaP with initial concentration of 10 mg l⁻¹ in 3 and 12 days of incubation, respectively. Addition of saccharides and low molecular weight polycyclic aromatic hydrocarbons appeared to have effect on the degradation ability, in particularly the addition of lactose and naphthalene. Furthermore, we demonstrated that lipidic particles could be observed in the presence of benzo[a]pyrene based on the morphologic performance of Aspergillus sp. BAP14 through scanning electronic microscopy (SEM) and atomic force microscopy (AFM), respectively.     

Benzo[a]pyrene metabolism in rat fetal hepatocytes culture. Improved methodology and effect of substrate concentration

The different characteristics of benzo[a[pyrene (BP) metabolism in primary fetal rat liver cell culture have been investigated. We have determined the extent of the in vivo [3H]BP metabolism by measuring all of the metabolites retained in the cell and excreted into the culture medium. The extent of the conjugation as well as the nature of the conjugates was established and the pattern of these metabolites analyzed by high performance liquid chromatography (HPLC). The fetal hepatocytes very actively metabolize BP and readily excrete in the culture medium all the produced metabolites in the form of sulfate and glucuronide conjugates. The relative proportion of those compounds varies as a function of the substrate concentration added to the cell culture, the higher the BP concentration, the more glucuronide conjugates. The HPLC analysis of the metabolites shows that BP-1,6-quinone and -3,6-quinone are the major excreted products, indicating the probable existence of an active 6 hydroxylation reaction in the fetal hepatocytes. On the other hand, the pattern of the different metabolites is influenced by the BP concentration. At low BP doses (0.8 microM), the relative amount of polar metabolites is twice as high and that of primary phenols twice as low, when compared to those produced by cells treated with 80 microM BP. The AHH activity drastically modifies the overall rate of the BP metabolism but does not affect the qualitative pattern of the excreted metabolites. The overall metabolism of [3H]BP by the cell culture can easily be estimated by measuring the release of the tritiated water from the substrate into the culture medium.     

Benzo[a]pyrene diol-epoxides: different mutagenic efficiency in human and bacterial cells

Monolayer cultures of diploid human fibroblasts and suspensions of S. typhimurium TA100 cells were treated with [3H]-labelled enantiomeric forms of benzo[a]pyrene anti and syn 7,8-dihydrodiol 9,10-epoxides. In both cell types, all of the enantiomers induced the formation of mutant 6-thioguanine (human) or 8-azaguanine-(bacterial)resistant cells. Diol-epoxide-modified nucleosides from human and from bacterial DNA hydrolysates were characterized by HPLC and showed essentially the same adduct species for human and bacterial cells treated with the same enantiomers. There were substantial differences, however, in the efficiency with which structurally-different adduct species were converted to mutant genotypes. In human cells, the mutagenic efficiency (mutation frequency/unit modified DNA) of the respective adduct species (+ anti much greater than -anti = +/- syn) at the hprt locus was exactly the opposite of that seen at a similar gene locus (gpt) in TA100 (-anti = +/- syn greater than + anti). The results suggest that the structural configuration of adducts in genomic DNA is important in determining whether a mutant genotype will result, and likewise, that there are differences in specificity between the human and bacterial systems which process these adduct lesions.     

Degradation of Benzo[a]pyrene by the Litter-Decomposing Basidiomycete Stropharia coronilla: Role of Manganese Peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn²⁺) supplied at a concentration of 200 μM stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn²⁺ than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn²⁺. Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter⁻¹ was converted in an in vitro reaction solution containing 1 U of MnP ml⁻¹ within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter⁻¹) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).     

Factors Influencing the Photodynamic Action of Benzo[a]pyrene on Escherichia coli

Death of Escherichia coli resulted when a buffer suspension was exposed simultaneously to colloidal benzo[a]pyrene (BP) and 355-mmu illumination. Neither hydrocarbon nor illumination alone caused death; oxygen had to be present. The survival curve had a shoulder, and then death proceeded exponentially with time. Death rate was independent of temperature between 6 and 32 C. The duration of the shoulder, however, decreased slightly with increase in temperature. The shoulder was not due to delay in BP entering the cell. Death was influenced by the composition of the medium in which the cells were grown prior to illumination. The amount of BP bound to the cells was determined after three ethyl alcoholether extractions. Appreciable binding occurred in the presence of 355-mmu illumination with air, and relatively little binding occurred under nitrogen; very little binding occurred in the dark with nitrogen or air. At the outset, rate of binding under illumination with air was not temperature-dependent, but with time it became strongly temperature-dependent. Binding under illumination with nitrogen was temperature-independent. Bound BP was associated primarily with cell protein. Cells in growth medium resisted death and BP binding. At 21 and 32 C, deoxyribonucleic acid damage occurred during exponential death. No damage was detected at 21 and 32 C in the dark with BP, under illumination in absence of BP, or under illumination with BP in a nitrogen atmosphere.     

Benzo[a]pyrene exposed to solar-simulated light inhibits apoptosis and augments carcinogenicity

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread environmental pollutants and several lines of experimental evidence have suggested a role in carcinogenesis. PAHs in the environment are exposed to sunlight and photomodified PAHs have been detected in contaminated sediment and air particulate matter; however, the carcinogenicity of photomodified PAHs is not well understood. In this study, we found that solar-simulated light-irradiated BaP (LBaP) inhibited apoptosis, leading to cancer. LBaP suppressed apoptosis induced by cell detachment and serum depletion in a dose and light-irradiated time-dependent manner. The antiapoptotic effect was related to the production of reactive oxygen species from degraded BaP. The cells that survived apoptosis by LBaP treatment were transformed having the ability to form colonies in soft agar and tumors in nude mice. These capabilities were specific to LBaP, not BaP itself. The results suggested that the carcinogenicity of PAHs may be attributable not only to the genetic damage induced by their metabolites, but also to the antiapoptotic effects of oxidative products on exposure to sunlight.     

Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate

Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.     

Benzo[a]pyrene metabolism and induction of enzyme-altered foci in regenerating rat liver

The metabolism of benzo[a]pyrene (BP) in regenerating rat liver and the induction of enzyme-altered foci (EAF) in the liver of partially hepatectomized rats, treated with BP and promoted with 2-acetylaminofluorene (2-AAF)/CCl4 was investigated. The aim was to examine factors that might be of importance for the tumorigenicity of BP in the regenerating rat liver, such as cytochrome P-450 activity and glutathione levels. In regenerating rat liver, obtained 18 h after partial hepatectomy (PH), the amount of microsomal cytochrome P-450 was reduced by 20% whereas the level of glutathione was elevated by 15% and the cytosolic glutathione transferase activity towards chlorodinitrobenzene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE) was unaffected. Microsomes from these animals had a reduced capacity to activate (-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BPD) to DNA-binding products but the pattern of BP metabolites was similar to that observed with control rat liver microsomes. Treatment of rats with 3-methylcholanthrene (MC, 50 mg/kg body wt.) increased cytochrome P-450 levels and glutathione transferase activity towards both substrates. Regenerating livers from these animals retained their cytochrome P-450 level and enzymatic activity towards BP and BPD. Regenerating rat liver microsomes from MC-treated animals were about 35 times more efficient in activating BPD than microsomes from uninduced, partially hepatectomized animals. Intraperitoneal administration of BP (50 mg/kg body wt.) 18 h after PH induced EAF in rats subsequently promoted with 2-AAF/CCl4. Pretreatment of rats with MC 66 h before PH and 84 h before BP administration, increased the number of EAF. In accordance with results by Tsuda et al. (Cancer Res., 40 (1980) 1157-1164), these studies demonstrate that BP is tumorigenic in regenerating rat liver, despite a reduced ability of the liver to activate this compound. Furthermore, MC, an inducer of certain cytochrome P-450 species ("aryl hydrocarbon hydroxylase"), potentiates the effect of BP.     

Benzo[a]pyrene-collagen interactions

In vitro interactions of benzo[a]pyrene (BaP) with acid-soluble type I collagen from rat tail tendon have been investigated. The fluorescence of BaP increases in the presence of collagen. Bound BaP inhibits the formation of collagen fibrils in solution. When BaP-collagen complexes are irradiated in air with UV (365 nm) light, BaP rapidly undergoes photooxidation with the further inhibition of fibril formation. Viscosity and circular dichroism (CD) studies show that neither BaP nor further UV-irradiation alters the size or helical conformation of the protein. During thermal denaturation of collagen, BaP fluorescence changes. Collagen from young rat tail tendon shows a pronounced drop at about 38 degrees C, whereas that from old rat tail tendon exhibits an increase with a plateau in the same temperature range. These anomalous changes are observed when tyrosine residues, present only in the non-helical terminal telopeptides of collagen, are excited at 275 nm, but not by direct BaP excitation at 387 nm. These findings suggest that the specific hydrophobic telopeptide region, which plays an important role in fibril formation, are affected by bound BaP.     

Benzo[a]pyrene diol epoxide-deoxyguanosine adducts are accurately bypassed by yeast DNA polymerase zeta in vitro

The possible role of bypass DNA polymerase zeta in mutagenic translesion synthesis past benzo[a]pyrene (BP) 7,8-diol-9,10-epoxide (DE) N(2)-deoxyguanosine (dG) adducts has been examined. We prepared 59-mer DNA templates containing dG adducts derived from trans opening of enantiomers of BP DE-2, in which the 7-hydroxyl group and epoxide oxygen are trans. The 10S-BP DE-dG and 10R-BP DE-dG adducts derive from the (+)- and (-)-DE-2 enantiomers, respectively. The adducted dG is located at a site identified as a G-->T mutational hotspot in random mutagenesis studies of (+)-BP DE-2 in Chinese hamster V-79 cells. Yeast pol zeta (complex of Gst-Rev3p and Rev7p) formed extension products (total of all lengths) of 71, 74 and 88% of a primer annealed to the 10S-BP DE-dG, 10R-BP DE-dG and non-adducted 59-mer templates, respectively. However, only 18 and 19% of the primer was extended to the full-length product on 10S-BP DE-dG and 10R-BP DE-dG adducted templates compared to 55% of the primer on the non-adducted template. A major 34-mer product corresponding to primer elongation up to and including the base before the adduct indicated that nucleotide incorporation opposite both adducts was strongly blocked. Full-length products were isolated from gels and subjected to PCR amplification and cloning. Sequence analysis of more than 300 clones of these full-length products on each template showed that only the correct dCMP was incorporated opposite both the adducted and non-adducted G-hotspot in the template. This corresponds to a probability of mutation lower than 0.3%, the limit of detection, and demonstrates the remarkable fidelity of yeast pol zeta in translesion synthesis past these BP DB-dG lesions in vitro.     

Early-Life Exposure to Benzo[a]pyrene Increases Mutant Frequency in Spermatogenic Cells in Adulthood

Children are vulnerable to environmental mutagens, and the developing germline could also be affected. However, little is known about whether exposure to environmental mutagens in childhood will result in increased germline mutations in subsequent adult life. In the present study, male transgenic lacI mice at different ages (7, 25 and 60 days old) were treated with a known environmental mutagen (benzo[a]pyrene, B[a]P) at different doses (0, 50, 200 or 300 mg/kg body weight). Mutant frequency was then determined in a meiotic cell type (pachytene spermatocyte), a post-meiotic cell type (round spermatid) and epididymal spermatozoa after at least one cycle of spermatogenesis. Our results show that 1) mice treated with B[a]P at 7 or 25 days old, both being pre-adult ages, had significantly increased mutant frequencies in all spermatogenic cell types tested when they were 60 days old; 2) spermatogenic cells from mice treated before puberty were more susceptible to B[a]P-associated mutagenesis compared to adult mice; and 3) unexpectedly, epididymal spermatozoa had the highest mutant frequency among the spermatogenic cell types tested. These data show that pre-adult exposure to B[a]P increases the male germline mutant frequency in young adulthood. The data demonstrate that exposure to environmental genotoxins at different life phases (e.g., pre-adult and adult) can have differential effects on reproductive health.     

A Common Carcinogen Benzo[a]pyrene Causes Neuronal Death in Mouse via Microglial Activation

Background

Benzo[a]pyrene (B[a]P) belongs to a class of polycyclic aromatic hydrocarbons that serve as micropollutants in the environment. B[a]P has been reported as a probable carcinogen in humans. Exposure to B[a]P can take place by ingestion of contaminated (especially grilled, roasted or smoked) food or water, or inhalation of polluted air. There are reports available that also suggests neurotoxicity as a result of B[a]P exposure, but the exact mechanism of action is unknown.

Methodology/Principal Findings

Using neuroblastoma cell line and primary cortical neuron culture, we demonstrated that B[a]P has no direct neurotoxic effect. We utilized both in vivo and in vitro systems to demonstrate that B[a]P causes microglial activation. Using microglial cell line and primary microglial culture, we showed for the first time that B[a]P administration results in elevation of reactive oxygen species within the microglia thereby causing depression of antioxidant protein levels; enhanced expression of inducible nitric oxide synthase, that results in increased production of NO from the cells. Synthesis and secretion of proinflammatory cytokines were also elevated within the microglia, possibly via the p38MAP kinase pathway. All these factors contributed to bystander death of neurons, in vitro. When administered to animals, B[a]P was found to cause microglial activation and astrogliosis in the brain with subsequent increase in proinflammatory cytokine levels.

Conclusions/Significance

Contrary to earlier published reports we found that B[a]P has no direct neurotoxic activity. However, it kills neurons in a bystander mechanism by activating the immune cells of the brain viz the microglia. For the first time, we have provided conclusive evidence regarding the mechanism by which the micropollutant B[a]P may actually cause damage to the central nervous system. In today''s perspective, where rising pollution levels globally are a matter of grave concern, our study throws light on other health hazards that such pollutants may exert.     

Comparative Sorption of Phenanthrene and Benzo[α]pyrene to Soil Humic Acids

Sorption of phenanthrene (Phen) and benzo[α]pyrene (BaP) by humic acids (HAs) extracted from four typical soils in China, including cinnamon soil, fluvo-aquic soil, red soil, and mountain meadow soil were investigated. All sorption data were fitted well by the Freundlich model, but BaP exhibited stronger and more nonlinear sorption than Phen by a given HA. For Phen isotherms, there was a positive relation between K _oc values and aliphaticity of HAs, whereas a negative correlation was observed between n values and aliphaticity. This indicated the importance of aliphatic groups in Phen sorption capacity and nonlinearity. Compared to Phen, a similar trend was obtained between n values of BaP and aliphaticity or aromatic carbon. However, no correlation existed between K _oc values for BaP and aliphaticity or aromatic carbon.